Differential Retention of Gene Functions in a Secondary Metabolite Cluster Hannah T. Reynolds,‡,1 Jason C. Slot,*,1 Hege H. Divon,2 Erik Lysøe,3 Robert H. Proctor,4 and Daren W. Brown*,4 1Department of Plant Pathology, The Ohio State University, Columbus, OH 2Norwegian Veterinary Institute, Oslo, Norway 3Department of Biotechnology and Plant Health, NIBIO – Norwegian Institute of Bioeconomy Research, A˚s, Norway 4Mycotoxin Prevention and Applied Microbiology Research Unit, U.S. Department of Agriculture†, Agricultural Research Service, Peoria, IL †Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer. ‡Present address: Department of Biological and Environmental Sciences, Western Connecticut State University, Danbury, CT. Downloaded from https://academic.oup.com/mbe/article/34/8/2002/3778301 by guest on 28 September 2021 *Corresponding authors: E-mails: [email protected]; [email protected]. Associate editor: Jeffrey Townsend Abstract In fungi, distribution of secondary metabolite (SM) gene clusters is often associated with host- or environment-specific benefits provided by SMs. In the plant pathogen Alternaria brassicicola (Dothideomycetes), the DEP cluster confers an ability to synthesize the SM depudecin, a histone deacetylase inhibitor that contributes weakly to virulence. The DEP cluster includes genes encoding enzymes, a transporter, and a transcription regulator. We investigated the distribution and evolution of the DEP cluster in 585 fungal genomes and found a wide but sporadic distribution among Dothideomycetes, Sordariomycetes, and Eurotiomycetes. We confirmed DEP gene expression and depudecin production in one fungus, Fusarium langsethiae. Phylogenetic analyses suggested 6–10 horizontal gene transfers (HGTs) of the cluster, including a transfer that led to the presence of closely related cluster homologs in Alternaria and Fusarium. The analyses also indicated that HGTs were frequently followed by loss/pseudogenization of one or more DEP genes. Independent cluster inactivation was inferred in at least four fungal classes. Analyses of transitions among functional, pseudogenized, and absent states of DEP genes among Fusarium species suggest enzyme-encoding genes are lost at higher rates than the transporter (DEP3)andregulatory(DEP6) genes. The phenotype of an experimentally-induced DEP3 mutant of Fusarium did not support the hypothesis that selective retention of DEP3 and DEP6 protects fungi from exogenous depudecin. Together, the results suggest that HGT and gene loss have contributed significantly to DEP cluster distribution, and that some DEP genes provide a greater fitness benefit possibly due to a differential tendency to form Article network connections. Key words: secondary metabolites, gene cluster, phylogenetic analysis, pseudogenization, horizontal gene transfer, depudecin. Introduction proteins that export the SM out of the cells in which it is synthesized, and a transcription factor that activates expres- Metabolites that provide a selective advantage in certain en- sion of the cluster genes. vironments but otherwise are not essential for growth, devel- Recent studies in fungal genome evolution have provided opment or reproduction are often referred to as secondary evidence that horizontal gene transfer (HGT) of SM biosyn- metabolites (SMs). The evolutionary history of SMs can be thetic gene clusters plays a significant role in fungal ecological dynamic because of shifting selection pressures that result adaptation (Walton 2000; Jargeatetal.2003; Bain et al. 2007; from environmental changes and co-evolution of species Fedorova et al. 2008; Khaldi et al. 2008; Slot and Rokas 2011; that interact via SMs (Stone and Williams 1992; Challis and Campbell et al. 2013; Chen et al. 2013; McGary et al. 2013; Hopwood 2003; Wink 2003). In bacteria and fungi, genes Proctor et al. 2013; Schumacher et al. 2013; Greene et al. 2014; encoding related metabolic functions are frequently found Brown et al. 2015). These observations are consistent with the in clusters (Brown et al. 1996, 2015; Bangera and “selfish cluster/operon” model of gene cluster evolution Thomashow 1999; Fischbach et al. 2008; Slot and Rokas (Lawrence and Roth 1996; Lawrence 1999). Under this model, 2011; Takos and Rook 2012; Campbell et al. 2013; Staehlin genes sharing a common function (e.g., a biosynthetic path- et al. 2016). Such clusters typically include genes coding for way) have a higher probability of persisting when they are enzymes that catalyze synthesis of the SM(s), transport horizontally transferred together in a cluster compared with Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2017. This work is written by US Government employees and is in the public domain in the US. 2002 Mol. Biol. Evol. 34(8):2002–2015 doi:10.1093/molbev/msx145 Advance Access publication April 28, 2017 Differential Retention of Gene Functions . doi:10.1093/molbev/msx145 MBE when they are transferred individually, because individual Fusarium genome dataset. Hypotheses for the role of selec- cluster genes cannot confer a selectable function alone. tion in the evolutionary mode of the DEP cluster, including Thus, organization of genes into a cluster benefits the genes the preservation of DEP function following HGT, and the themselves. Selfish clustering is expected to preferentially ben- differential retention of DEP3 and DEP6, were tested using efit genes in clusters responsible for synthesis of SMs under experimental approaches in Fusarium. weak or transient selection, because if the genes were not clustered they would be less likely to be retained in a genome Results through vertical inheritance. A gene cluster thought to have a weakly selected function Distribution and Clustering of DEP Genes is responsible for synthesis of the SM depudecin, a small linear We surveyed the presence of DEP genes in phylogenetically polyketide originally isolated from the fungus Alternaria bras- diverse fungi by BLAST analysis using nucleotide sequences or sicicola (Dothideomycetes, Ascomycota) that acts as a his- predicted amino acid sequences of the six A. brassicicola DEP tone deacetylase (HDAC) inhibitor (Matsumoto et al. 1992). genes as queries against 585 genomes and the Non- Downloaded from https://academic.oup.com/mbe/article/34/8/2002/3778301 by guest on 28 September 2021 In plants, HDACs and histone acetyltransferases play an inte- redundant sequence database at NCBI. These analyses gral role in modifying regulation of gene expression and affect led to the identification of DEP gene homologs in 120 a variety of cellular and developmental processes (Hollender genomes of species in five classes of the Ascomycota: and Liu 2008). Depudecin makes a small contribution to The Dothideomycetes, Eurotiomycetes, Leotiomycetes, pathogenesis; depudecin-non-producing mutants of A. bras- Sordariomycetes, and Lecanoromycetes (see supplemen- sicicola exhibit a 10% reduction in virulence on cabbage tary figs. S1–S6, Supplementary Material online, table 1 (Wight et al. 2009). In contrast, the HDAC inhibitor HC toxin and see supplementary table S1, Supplementary Material makes a more substantial contribution to virulence of online). In preliminary phylogenetic analyses, DEP1 and Cochliobolus carbonum (Dothideomycetes) on maize DEP3–DEP5 were found in large, multigene families. (Walton 2006). Depudecin also has moderate antiprotozoan Sequences in these families were manually removed at activity against parasites such as Plasmodium (Isaka et al. strongly supported nodes in order to improve the evalu- 2000)andNeospora (Kwon et al. 2003). However, it requires ation of homologs most closely related to the DEP genes. a markedly higher concentration (500 ng/ml; 0.47 mM) to in- Sequences were removed from the DEP3 gene family, hibit Neospora cell replication to the same degree as apicidin which had the highest number of homologs, so that it (15 ng/ml; 0.02 mM), another HDAC inhibitor. Thus depude- included all taxa containing five or more DEP homologs cin is expected to provide a weak and transient benefit to based on the other DEP gene trees (see supplementary fungi compared with more potent HDAC inhibitors, and to figs. S1–S6, Supplementary Material online). At least one be under inconsistent selection. DEP gene sequence was found in the 120 genomes, while 95 The depudecin biosynthetic gene (DEP) cluster consists of DEP3 homologs were distributed among 15 Dothideomycete, six adjacent and coregulated genes, DEP1–DEP6 (Wightetal. 15 Eurotiomycete, 1 Lecanoromycete, 1 Leotiomycete, and 63 2009). Three genes encode enzymes: DEP5 encodes a polyke- Sordariomycete genomes (see supplementary table S1, tide synthase (PKS), and DEP2 and DEP4 encode monooxy- Supplementary Material online, figs. 1 and 2). genases. The PKS is predicted to synthesize the carbon Multiple DEP cluster homologs exhibited gene rearrange- backbone of depudecin, while the monooxygenases likely ox- ment and loss (fig. 1, table 2). The gene order in A. brassicicola ygenate the backbone. DEP3 encodes a Major Facilitator (DEP1, DEP2, DEP3, DEP4, DEP5, DEP6) was present only in a Superfamily (MFS) transporter, DEP6 encodes a Zn2Cys6 group of Dothideomycete and Sordariomycete species that DNA-binding transcription factor (C6) and DEP1 encodes a were resolved into one clade in the DEP3 gene